This proposal to facilitate research career development will provide the applicant with skills necessary to apply a comprehensive, hierarchical approach to study the role of angiotensin II in brown adipose tissue thermogenesis. Previous studies in our laboratory have demonstrated angiotensinogen messenger RNA expression in brown adipose tissue of the rat. The working hypothesis of the proposed research is that endogenously produced angiotensin II in brown adipose tissue acts to regulate thermogenesis via modulation of sympathetic neurotransmission. The following comprehensive approach will be used to address the working hypothesis: 1) determine processing and secretion of angiotensinogen in isolated brown adipocytes using pulse-chase protocols coupled with western blot for angiotensinogen; 2) examine the cellular localization and subtype predominance of angiotensin II receptors in isolated brown adipocytes, synaptosomes, and stromal/vascular elements of brown adipose tissue; 3) determine the processing enzymes involved in production of angiotensin II in brown adipose tissue by using a) immunohistochemistry for enzyme localization, b) inhibitors of the renin-angiotensin system and effects on angiotensin II production, c) surgical denervation to examine if angiotensin II acts as a neuropeptide, and d) polymerase chain reaction using oligo's constructed to aspartyl protease portions of the renin gene to isolate alternative enzymes involved in processing to angiotensin II; 4) determine if endogenous angiotensin II regulates stimulated thermogenesis by examining the brown adipose renin-angiotensin system after cold- and diet-induced thermogenesis as well as examining the effect of the angiotensin II receptor antagonist, DuP 753, on stimulated thermogenesis. As evidence of Institutional support of this endeavor, the applicant will be relieved of teaching and committee assignments, will be provided with funds to support a postdoctoral research fellow, and work time necessary to learn techniques with collaborative investigators on receptor binding, immunohistochemistry, and polymerase chain reaction. Collectively, these studies will permit a rigorous examination of the role of angiotensin II in thermogenesis of brown adipose tissue. Additionally, as the newly acquired skills are appropriate for the study of other contemporary molecular and cellular processes, the applicant will be able to apply a comprehensive research approach in future studies examining alterations in brown adipose tissue thermogenesis in obesity.